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Home , Aminopterin, Capecitabine, Epirubicin, Ixabepilone, Streptozotocin, Taxane

Drugs (2015) 75:1993–2016 DOI 10.1007/s40265-015-0489-4

REVIEW ARTICLE

Recent Advances in the Development of Antineoplastic Agents Derived from Natural Products

Matthew Trendowski1

Published online: 26 October 2015 Ó The Author(s) 2015. This article is published with open access at Springerlink.com

Abstract Through years of evolutionary selection pres- sures, organisms have developed potent toxins that coin- Key Points cidentally have marked antineoplastic activity. These natural products have been vital for the development of Natural products have potentiated many novel drug multiagent treatment regimens currently employed in classes employed in cancer , including cancer chemotherapy, and are used in the treatment of a mechanistic target of rapamycin inhibitors, protein variety of malignancies. Therefore, this review catalogs synthesis inhibitors, nucleic acid-directed agents, recent advances in -based and -directed agents via the examination of mechanisms of action and available Recent advances in immunotherapy have enabled clinical data to highlight the utility of these novel com- highly cytotoxic natural products to be targeted pounds in the burgeoning age of precision medicine. The towards specific tissues review also highlights the recent development of antibody- There are still many natural products with drug conjugates and other immunotoxins, which are cap- mechanisms not currently seen in the clinical setting able of delivering highly cytotoxic agents previously that could be very beneficial to the field of oncology deemed too toxic to elicit therapeutic benefit preferentially to neoplastic cells. Finally, the review examines natural products not currently used in the clinic that have novel mechanisms of action, and may serve to supplement cur- rent chemotherapeutic protocols. 1 Introduction

The diversity of natural products currently used in the clinical setting to treat solid tumors, as well as dissemi- nated cancers is truly extensive. Under the pressure of natural selection, various species produce cytotoxic sec- ondary metabolites to combat potential predators, prey, or competition in the so-called ‘‘arms race’’ of evolution. Remarkably, some of these natural toxins appear to exhibit potent antineoplastic activity, and after years of research, have found their way from the ocean or soil to the highly heterogeneous environment of clinical oncology. The ori- & Matthew Trendowski gins of cancer chemotherapy can be traced to human-made [email protected] compounds, as Goodman, Gilman, and colleagues at Yale 1 Department of Biology, Syracuse University, 107 College University began investigating the potential of nitrogen Place, Syracuse, NY 13244, USA mustards in 1942 [1], which was shortly followed by 1994 M. Trendowski

Sidney Farber’s use of the to induce review provides an extensive coverage of novel natural remissions among children with in 1947 [2, 3]. product-based antineoplastic agents, additional agents have However, the institution of natural products and semisyn- seen recent success in the clinical setting, and the reader is thetic derivatives of these compounds in the latter part of referred to the following reviews for further information the 20th century potentiated the idea of concomitant [4–6]. In addition, the diversity of natural product-based chemotherapy; using a variety of antineoplastic agents with antineoplastic agents and their derivatives currently different mechanisms of action to significantly perturb approved by the US Food and Drug Administration (FDA) neoplastic development, and in some cases, produce long- are highlighted in Table 1. They serve as a reminder of term remissions. how important nature has been in the treatment of many, if Owing to recent advances in molecular biology, inves- not most types of malignancy. tigators have begun unraveling essential oncogenic path- ways in carcinogenesis, potentiating an era of chemotherapy in which it is possible to theorize cancer- 2 Microtubule-Disrupting specific targets. This has launched the introduction of precision medicine in cancer chemotherapy in which Eribulin is a fully synthetic, macrocyclic ketone analog of clinicians now have the capability of selecting optimal the marine sponge natural product halichondrin B (Fig. 1), therapies based on the genetic and phenotypic profile of the a potent antimitotic initially isolated in 1986 from Hali- patient’s malignancy in addition to traditional broad- chondria okadai [7]. Although halicondrin B was desig- spanning cytotoxic antineoplastic intervention. Despite nated for preclinical development after it was found to be these commendable advances in targeted therapy, natural highly cytotoxic against murine leukemia cells, difficulty products and their derivatives are still extensively relied in collecting sufficient material for developmental studies upon against malignancies where finding cancer-specific slowed its progress, and interest began to fade. However, targets has been less successful, and are often used in the discovery that halocondrin B activity resides in the combination with these targeted approaches to generate macrocyclic lactone C-1 to C-38 moiety [8] paved the way more thorough treatment protocols. Further, novel natural for development of a simplified synthetic analog, culmi- product derivatives have shown notably efficacy against nating in the design of eribulin. previously unresponsive malignancies at the clinical level, As with , eribulin exerts its cytotoxic suggesting that natural product-based drug discovery still effects by interfering with microtubule dynamics, and has considerable utility in the burgeoning era of personal- inhibiting polymerization [9, 10]. In addition, eribulin ized chemotherapy. Finally, natural products have the also works through an end-poisoning mechanism, result- potential to improve novel immunotherapeutic strategies ing in the inhibition of microtubule growth, and even by conjugating monoclonal antibodies (mABs) or cytoki- sequesters tubulin into nonfunctional aggregates, pro- nes to highly cytotoxic compounds that have too low of a moting G2/M phase arrest and apoptosis [11]. However, without an appropriate guidance the two drug classes contrast in that eribulin does not bind mechanism. the sides of tubulin polymers, and therefore does not This review catalogs recent advances in natural product markedly potentiate depolymerization [12, 13]. It does drug discovery that have potentiated promising activity suppress spindle microtubule tension by interfering with against aggressive malignancies, and have enabled a more centromere dynamics, as seen with some vinca alkaloids precise delivery of highly cytotoxic, natural product-based (particularly and vinflunine), but does so by agents to reduce unintended side effects. Specifically, this inhibiting relaxation rates and the time spent stretching review covers the commendable advances in the develop- and relaxing, without the corresponding suppressive ment of microtubule-directed agents (eribulin and epothi- effects on stretching rates observed with the other agents lones), mechanistic target of rapamycin (mTOR) inhibitors [14]. Further, eribulin inhibits tubulin polymerization by (everolimus and temsirolimus), protein synthesis inhibitors binding the interdimer interface or the b-tubulin subunit (omacetaxine mepesuccinate), nucleic acid-directed agents alone contrary to other microtubule-directed agents, (), engineered cytokine proteins (denileukin including and [12]. Interestingly, diftitox), and antibody-drug conjugates (ADCs; brentux- eribulin demonstrates significant activity against bIII- imab vedotin, , calicheamicin con- tubulin, an isotype that is overexpressed in cells resistant jugated monoclonal antibodies, and exotoxin conjugates). to microtubule inhibitors [15, 16], indicative of its unique In addition, the review will highlight several novel natural clinical utility. products that act by mechanisms not currently seen in the In regard to its clinical pharmacologic profile, eribulin clinic (cytochalasins and withanolides) to address their demonstrates linear with rapid systemic potential utility in cancer chemotherapy. Although this distribution, but has a t1/2 of 40 h and around 49–65 % Natural Product-Based Antineoplastic Agents 1995

Table 1 US Food and Drug Administration (FDA) approved uses of natural products in cancer chemotherapy Agent Drug classification/species of origin Mechanism of action FDA approved use

Omacetaxine Inhibits protein synthesis and is Chronic- or accelerated-phase CML with mepesuccinate Cephalotaxus harringtonia independent of direct Bcr-Abl binding resistance and/or intolerance to two or (SynriboÒ) more TKIs Topo II inhibitor, intercalating agent Remission induction in adult AML or in (Cerubidine) peucetius both children and adults for ALL DaunoXomeÒ Citrate liposome formulation Advanced HIV-associated Kaposi’s sarcoma Anthracycline Topo II inhibitor, intercalating agent ALL, AML, Wilms tumor, Ò (Adriamycin ) Streptomyces peucetius neuroblastoma, soft tissue and bone sarcoma, breast, ovarian, thyroid, bronchiogenic, gastric and transitional cell bladder carcinomas, HL, NHL DoxilÒ Liposome formulation AIDS-related Kaposi’s sarcoma, ovarian carcinoma, multiple myeloma Anthracycline Topo II inhibitor, intercalating agent Axillary node-positive breast carcinoma Ò (Ellence ) Streptomyces peucetius Anthracycline Topo II inhibitor, intercalating agent Adults with AML classified M1 to M7 Ò (Idamycin ) Streptomyces peucetius (French-American-British system) Anthracycline Topo II inhibitor, intercalating agent Carcinoma in situ of the urinary bladder Ò (Valstar ) Streptomyces peucetius Anthracycline Topo II inhibitor, intercalating agent Adult AML, symptomatic - (NovantroneÒ) Streptomyces peucetius refractory prostate adenocarcinoma Brentuximab Antibody-drug conjugate, dolastatin, MMAE enters cells expressing CD30, HL after failure of ASCT or after failure vedotin MMAE is derived from peptides potentiating microtubule inhibition in of two prior multiagent (AdcetrisÒ) found in Dolabella auricularia addition to the antineoplastic effects of chemotherapeutic regimens in those brentuximab who are not ASCT candidates, sALCL Trastuzumab Antibody-drug conjugate, macrolide, DM1 enters cells expressing HER2/neu HER2? breast carcinoma, metastatic emtansine DM1 is derived from maytansine, receptor, potentiating microtubule gastric or gastroesophageal (KadcylaÒ) which can be extracted from plants inhibition in addition to the adenocarcinoma with HER2 of the genus Maytenus antineoplastic effects of trastuzumab overexpression Mitomycin Alkylating agent, crosslinks DNA Disseminated gastric adenocarcinoma, Streptomyces caespitosus or disseminated pancreactic Streptomyces lavendulae adenocarcinoma Topo I inhibitor Metastatic colorectal carcinoma Ò (Camptosar ) acuminata Camptothecin Topo I inhibitor Cervical carcinoma, metastatic ovarian Ò (Hycamtin ) Camptotheca acuminata carcinoma, SCLC Denileukin Engineered cytokine protein Composed of diphtheria toxin fragments Persistent or recurrent CTCL in patients diftitox Corynebacterium diphtheriae linked to IL-2 sequences, interacts with who express the CD25 component of (OntakÒ) IL-2 cell surface receptors before the IL-2 receptor inhibiting protein synthesis Depletes , an amino acid Component of a multiagent induction Ò (Elspar , Elspar: Escherichia coli required by some regimen for ALL ErwinaseÒ) Erwinase: Erwinia chrysanthemi PegasparagaseÒ Pegylated version Patient has hypersensitivity to asparaginase Stabilizes formed Metastatic or locally advanced breast (IxempraÒ) carcinoma after failure of an anthracycline and a Glycopeptide Unresolved, but does induce DNA strand Squamous cell carcinomas, NHL, Ò (Blenoxane ) Streptomyces verticillus breaks testicular cancers, HL, malignant pleural effusions Lignan Topo II inhibitor Testicular cancers, SCLC (VepesidÒ) peltatum 1996 M. Trendowski

Table 1 continued Agent Drug classification/species of origin Mechanism of action FDA approved use

Etoposide Ester derivative that increases water Testicular cancer, SCLC phosphate solubility (EtophosÒ) Lignan Topo II inhibitor Refractory childhood ALL (VumonÒ) Podophyllum peltatum Eribulin Macrolide Inhibits the growth phase of Metastatic breast carcinoma that has Ò (Halaven ) Halichondria okadai microtubules without affecting the received at least two prior shortening phase, sequesters tubulin chemotherapy regimens for late-stage into nonproductive aggregates disease, including both anthracycline- and taxane-based Everolimus mTOR inhibitor Inhibits mTOR by binding FKBP-12 Postmenopausal women with advanced Ò (Afinitor , Streptomyces hygroscopicus hormone receptor? and ER- breast Afinitor carcinoma, PNET, RCC, renal DisperzÒ) angiomyolipoma, pediatric and adult SEGA Temsirolimus mTOR inhibitor Inhibits mTOR by binding FKBP-12 Advanced RCC Ò (Torisel ) Streptomyces hygroscopicus Alkylating agent Metastatic islet cell carcinoma of the Ò (Zanosar ) Streptomyces achromogenes Polypeptide Binds DNA at the transcription initiation Wilms tumor, pediatric (CosmegenÒ) various species of the genus complex and prevents elongation of , Ewing’s sarcoma, Streptomyces RNA chain by RNA polymerase metastatic and nonseminomatous testicular cancer, gestational trophoblastic neoplasia, locally recurrent or locoregional solid malignancies Taxane Stabilizes formed microtubules Ovarian carcinoma, breast carcinoma, Ò (Taxol ) brevifolia NSCLC, AIDS-related Kaposi’s sarcoma AbraxaneÒ Protein bound, conjugated to albumin Metastatic breast carcinoma, locally advanced or metastatic NSCLC Taxane Stabilizes formed microtubules NSCLC, breast carcinoma, prostate Ò (Taxotere ) Taxus brevifolia adenocarcinoma, gastric adenocarcinoma, head and neck carcinomas Taxane Stabilizes formed microtubules Hormone-refractory prostate (JevtanaÒ) Taxus brevifolia adenocarcinoma Inhibits tubule polymerization Acute leukemias, HL, NHL, (OncovinÒ) roseus neuroblastoma, Wilms tumor, rhabdomyosarcoma MarqiboÒ Liposome formulation Philadelphia chromosome?ALL Vinca alkaloid Inhibits tubule polymerization Testicular cancers, HL, NHL, mycosis (VelbanÒ) fungoides, Kaposi’s sarcoma, histiocytic lymphoma, Letterer-Siwe disease (histiocytosis X), breast carcinoma, choriocarcinoma Vinorelbine Vinca alkaloid Inhibits tubulin polymerization NSCLC (NavelbineÒ) Catharanthus roseus Underline indicates different formulations of the agent. Bold indicates that the compound is a synthetic or semisynthetic derivative of the original natural product AIDS acquired human immunodeficiency syndrome, ALL acute lymphoid leukemia, AML acute myeloid leukemia, ASCT autologous stem cell transplant, CD cluster of differentiation, DM1 mertansine, CML chronic myeloid leukemia, CTCL cutaneous T-cell lymphoma, ER estrogen receptor, FKBP12 12 kDa FK506 binding protein, HIV human immunodeficiency virus, HL Hodgkin’s lymphoma, IL-2 interleukin-2, MMAE monomethyl auristatin E, mTOR mechanistic target of rapamycin, NHL non-Hodgkin’s lymphoma, NSCLC non-small-cell lung carcinoma, PNET neuroendocrine tumors of pancreatic origin, RCC renal cell carcinoma, sALCL systemic anaplastic large-cell lymphoma, SEGA subependymal giant-cell astrocytoma, SCLC small-cell lung carcinoma, Topo DNA topoisomerase, TKI tyrosine kinase inhibitor Natural Product-Based Antineoplastic Agents 1997

Fig. 1 Molecular diversity of antineoplastic agents derived from natural products. AMU atomic mass unit, DM1 mertansine, MW molecular weight, SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate remains protein bound in circulation [3, 17]. The majority undiluted or diluted in 100 mL of 0.9 % normal saline [17]. of the agent is eliminated through bile emulsification and Although this schedule is typically well tolerated, eribulin fecal . Eribulin is manufactured at 0.5 mg/mL is known to potentiate notable ([grade 3 tox- concentrations, and no routine premedication is needed. icity is *57 %), elevate transaminases, and induce One of the most common dosing schedules is 1.4 mg/m2 peripheral neuropathy (*8 % for grade 3 neuropathy and over 2–5 min on days 1 and 8 of a 21-day cycle, either 0.4 % for grade 4 neuropathy). 1998 M. Trendowski

Owing to the unique mechanisms by which eribulin overexpression, epothilones appear to be equally as potent inhibits microtubule dynamics, the agent is FDA approved against cells demonstrating this phenotype [29, 30]. for metastatic breast carcinoma refractory or relapsed on at Whereas paclitaxel is less effective in suppressing the least two prior treatment protocols for late-stage disease, growth rate and catastrophe frequency of purified a/bIII including both anthracycline- and taxane-based tubulin, ixabepilone markedly suppresses the dynamic chemotherapies [18]. This approval stems in large part instability of a/bIII dimers [31]. Further, ixabepilone has from a phase III open-label study (n = 762) in which exhibited substantially higher activity than either taxanes eribulin improved overall survival [median of 13.1 months, or vinca alkaloids against neoplastic cells in vitro and 95 % confidence interval (CI) 11.8–14.3] in comparison to in vivo in which taxane resistance is associated with bIII treatments of the physicians’ choice (median of overexpression [32–34]. In regards to its structure-activity 10.6 months, 9.3–12.5; hazard ratio = 0.81, 95 % CI relationship with tubulin, epothilones appear to bind the 0.66–0.99; p = 0.041) [19]. Peripheral neuropathy was the taxane pocket of b-tubulin and promote structuring of the most common adverse event leading to discontinuation M-loop into a short helix, as demonstrated by epothilone A from eribulin, occurring in 24 (5 %) of 503 patients. In [35]. Consequently, the M-loop establishes lateral tubulin addition, eribulin has also been investigated for use in a contacts in microtubules, thereby potentiating microtubule variety of other solid tumors, including non-small-cell lung assembly and stability. carcinoma, head and neck carcinoma, pancreatic adeno- Modifications to the macrolide ring have been shown to carcinoma, prostate adenocarcinoma, and various sarco- alter both the antineoplastic activity and pharmacologic mas. Published data indicate that non-small-cell lung properties of epothilones. Second- and third-generation carcinoma patients receive some benefit from eribulin, but congeners have been synthesized that possess higher the agent does not appear to be effective in the treatment of potency and enhanced water solubility compared with the head and neck or pancreatic malignancies [20]. In addition, original natural products [32, 33]. Ixabepilone, a second- eribulin appears to demonstrate activity against metastatic generation semisynthetic analog of epothilone B has castration-resistant prostate adenocarcinoma and advanced nitrogen substituted at position 16 of the macrolide ring soft-tissue sarcoma with a relatively favorable toxicity instead of oxygen, making it a lactam [34] (Fig. 1). This profile being observed [21, 22]. substitution increases water solubility and plasma stability in comparison to epothilones B and D, but also reduces cytotoxicity by one-fold [36]. In addition to ixabepilone, a 3 Microtuble-Stabilizing Epothilones second semisynthetic derivative of epothilone B, BMS- 310705, a congener synthesized by the substitution of a Epothilones are 16-member macrolide microtubule-stabi- hydroxyl group with an amino group at C-21 of the lizing agents initially isolated in 1987 from the So ce90 methylthiazole side chain, is 10-fold more water soluble strain of the myxobacterium Sorangium cellulosum [23, than epothilone B and is more cytotoxic than epothilone D 24]. Originally investigated for their antimycotic activity, in multiple human neoplastic cell lines [37, 38]. Several samples of epothilones A and B sent to the National Cancer other recent novel epothilone derivatives have gained Institute in 1994 demonstrated potent antineoplastic notable preclinical and clinical interest. 20-Desmethyl-20 activity in multiple cancer cell lines that was comparable methylsulfanyl epothilone B (ABJ-879) is a second-gen- and sometimes superior to paclitaxel. However, a critical eration derivative that exhibits more cytotoxicity than difference between epothilones and other bulky natural epothilone B [39]. Sagopilone (ZK-EPO) is the first fully products or derivatives is that overexpression of ATP- synthetic, third-generation epothilone B derivative that binding cassette (ABC) transporters does not significantly exhibits greater potency in vitro relative to the other epo- alter the cytotoxicity of epothilones, as these congeners thilones, retains activity in multidrug-resistant malignant have minimal substrate affinity for these proteins [25]. cells not observed in other congeners, and even crosses the Epothilones A and B have marked anti-proliferative blood–brain barrier, indicating the potential for penetration activity in neoplastic cells with elevated levels of perme- into the central nervous system [40]. The preclinical data ability glycoprotein (P-gp) [26, 27], and tumor samples have been so compelling that sagopilone has been inves- obtained from patients that respond to ixabepilone have tigated at the clinical level against advanced solid tumors shown significantly elevated levels of ABC transporters and melanoma, with the agent demonstrating favorable P-gp and multidrug resistance-associated protein 1 [27]. It pharmacokinetic data, a feasible toxicity profile, and anti- should be noted that multidrug-resistance protein 7 is tumor activity against melanoma not elicted by other capable of effluxing a variety of antineoplastic agents, congeners [41, 42]. Finally, a second-generation epothilone including epothilone B [28]. Although drug resistance to D analog, KOS-1584, exhibits 3- to 12-fold increased taxanes is often associated with b-III subunit potency compared with epothilones B and D, enhanced Natural Product-Based Antineoplastic Agents 1999 neoplastic tissue penetration, and reduced central nervous analogs of the macrolide rapamycin, a natural product system toxicity [43]. initially isolated from Streptomyces hygroscopicus in 1975 Despite the isolation and characterization of epothilones within the soils of Easter Island (also referred to as Rapa A–F, the only agent of this class to reach FDA approval is Nui, giving rise to the name of the agent) [55–57]. ixabepilone. Although ixabepilone has better aqueous sol- Although currently used as an immunosuppressive agent, ubility than paclitaxel, Kolliphor EL is still typically used rapamycin has also demonstrated marked cytostatic activ- as an excipient, and is contraindicated in patients who are ity against several cancer types [58]. Its unique pharma- hypersensitive to the vehicle [44, 45]. Most clinical cokinetic profile has prevented rapamycin from being investigations of ixabepilone have used a dosing schedule further developed as an antineoplastic agent, but its potent of 32–50 mg/m2 infused over 1 or 3 h on day 1 of a 21-day mechanisms of action have inspired the development of cycle. Nevertheless, a 3-h infusion time is recommended, temsirolimus, a novel soluble rapamycin derivative that has as more prominent neurotoxicity is often observed with a favorable toxicity profile in mammalian models, thereby shorter infusion times, and the FDA-approved dose and potentiating the use of mTOR inhibitors in chemotherapy. schedule is 40 mg/m2 intravenously (i.v.) over 3 h q3w As of now, only temsirolimus and the later developed [46]. Multiple phase I/II trials have revealed that ixabepi- analog everolimus have received FDA approval as anti- lone exposure is not significantly affected by patient neoplastic agents. characteristics (age, sex, renal function, body weight, body Rapamycin is a complex 21-member macrolide lactone surface area, race) [44, 47]. As with many mitotic inhibi- that contains a pipecolate moiety in the upper left region of tors, the most common grade 3 or 4 toxicity associated with the molecule. The agent inhibits mTOR primarily by standard single-agent dosing schedules is neutropenia crosslinking FKBP-12 (12 kDa FK506 binding protein) via (grade 3 neutropenia is observed in the range of 10–33 %, its methoxy functional group [59, 60]. The high affinity of while grade 4 neutropenia is observed in 7–32 % of rapamycin to FKBP-12 is mediated in part by its pipecolate patients, the wide range likely being a reflection of the region, which hydrogen bonds at two different hydrophobic number and type of prior therapies [46, 48–50]. binding pockets, as revealed by X-ray crystallography [61– Similar to eribulin, ixabepilone is currently indicated in 63]. Once bound, the rapamycin/FKBP-12 complex blocks metastatic or locally advanced breast carcinoma that is the binding of the accessory protein RAPTOR (regulatory- refractory or has relapsed on an anthracycline and a taxane, associated protein of mTOR) to mTOR, necessary for and is typically administered in combination with capeci- downstream phosphorylation of S6K1 and 4EBP1. Con- tabine [48, 50]. Interestingly, ixabepilone has shown sequently, S6K1 dephosphorylates, which reduces protein notable efficacy against triple-negative breast carcinoma synthesis and decreases cell motility and size [64–66]. In (TNBC), producing a pathologic complete response rate of addition, rapamycin induces dephosphorylation of 4EBP1. 26 % in TNBC patients in comparison to 15 % in the non- Such activity potentiates increases in p27, and decreases in triple-negative population [51]. Concomitant administra- cyclin D1 expression, invoking late blockage of G1/S tion of ixabepilone and has resulted in higher during the [66]. Although it is clear that rapa- overall response rates than monotherapy, and a phase III mycin induces apoptosis in neoplastic cells, the molecular trial of ixabepilone plus capecitabine produced a median mechanism of apoptosis has not yet been fully resolved. progression-free survival significantly longer for TNBC Temsirolimus has been an important addition to patients treated with ixabepilone plus capecitabine chemotherapeutic protocols indicated for renal cell carci- (4.2 months) in comparison to treatment with capecitabine noma (RCC) owing to the importance of mTOR in car- alone (1.7 months) [51]. In addition to breast carcinoma, cinogenesis; in RCC tumors, activated mTOR further ixabepilone has been investigated against non-Hodgkin’s exacerbates accumulation of HIF-1a (hypoxia-inducible lymphoma and pancreatic adenocarcinoma, both as a stand- factor 1-a) by increasing synthesis of this transcription alone agent and in concomitant chemotherapy [52–54]. factor and its angiogenic target gene products [67]. As such, rapamycin and its analogs have demonstrated notable antiangiogenic activity [68], indicating the poten- 4 Mechanistic Target of Rapamycin Inhibitors tial of combining these congeners with agents that elicit similar effects on neoplastic vasculature, including beva- Although medicinal chemists have made progress in per- cizumab, sorafenib, and sunitinib [69, 70]. In addition to its turbing the PI3K/AKT/mTOR (phosphatidylinositol-4,5- utility in RCC therapy, temsirolimus is being clinically bisphosphate 3-kinase/protein kinase B/mechanistic target evaluated against other carcinomas known to have elevated of rapamycin) pathway through the development of syn- mTOR activity, including malignancies of the breast and thetic compounds, the only mTOR inhibitors currently lung [71, 72]. Everolimus (Fig. 1) is an orally (p.o.) FDA approved for chemotherapeutic intervention are administered 40-O-(2-hydroxyethyl) derivative of 2000 M. Trendowski rapamycin that is currently indicated for postmenopausal ABL T315I-expressing LSCs in comparison to non-mutant women with advanced hormone receptor positive and BCR-ABL-expressing LSCs [82], indicating that the agent estrogen receptor negative breast carcinoma, as well as may be able to markedly inhibit clonal expansion in select neuroendocrine tumors of pancreatic origin, RCC, renal CML patients. angiomyolipoma, and both pediatric and adult Unlike most antineoplastic agents, omacetaxine mepe- subependymal giant cell astrocytoma [18]. The agent has succinate is administered subcutaneously (s.c.). with the also been clinically evaluated against gastric adenocarci- standard regimen being 1.25 mg/m2 s.c. b.i.d., days 1–14 noma, hepatocellular carcinoma, and in multiple types of every 28 days, with a maintenance treatment of 1.25 mg/m2 lymphoma [73–75]. s.c. b.i.d. for 7 days every 28 days [79]. Omacetaxine mepesuccinate disappears rapidly from plasma after ces-

sation of infusion, with an observed a-t1/2 of 5 h, a b-t1/2 of 5 Protein Synthesis Inhibitor Omacetaxine 9.3 h, and a mean steady-state terminal t1/2 of 7 h with Mepesuccinate biexponential decay observed. The agent undergoes a rapid metabolism with urinary excretion representing about Omacetaxine mepesuccinate (homoharringtonine) is a 12–15 % of the administered dose. In addition, omac- natural ester of the alkaloid cephalotaxine, a compound etaxine mepessucinate has a favorable toxicity profile with initially isolated and characterized in 1969 from Cepha- effects on and cardiovascular function being the most lotaxus harringtonia (Japanese plum yew) [76] (Fig. 1). prominent [79–81, 83]. Owing to its efficacy in TKI-re- Although cephalotaxine itself does not exhibit antineo- sistant cells, omacetaxine mepesuccinate is FDA approved plastic activity, fractionations of extracts obtained from for both chronic- and blast-phase CML [18], and clinical several variants of C. harringtonia produced a series of trials are currently ongoing to determine optimal agents to cephalotaxine esters that demonstrated antineoplastic use in combination with the protein synthesis inhibitor. activity. One of these compounds, homoharringtonine Interestingly, not as much interest has been paid towards (later renamed omacetaxine mepesuccinate), was shown to the potential of omacetaxine mepesuccinate in Philadelphia influence the progression of acute myeloid leukemia chromosome-positive ALL that is unresponsive to TKI (AML) and chronic myeloid leukemia (CML) in China therapy, and may be an avenue of future clinical interest. during the 1970s, with later studies in the confirming these findings [77, 78]. However, the clinical development of omacetaxine mepesuccinate was halted 6 Nucleic Acid-Directed Trabectedin after the development of imatinib, which has since shown remarkable activity in patients with Philadelphia chromo- Trabectedin is a novel antineoplastic agent that was iso- some positive CML and acute lymphoid leukemia (ALL). lated from the sea squirt Ecteinascidia turbinate in 1984 Nevertheless, reemergence in the investigation of omac- [84, 85], and has a relatively complex structure; three etaxine mepesuccinate quickly resumed once it was real- moieties, eight rings including one ized that a subset of indicated leukemias are either 10-membered heterocyclic ring containing a refractory or develop resistance to imatinib or related tyr- residue, and seven chiral centers (Fig. 1). While the E. osine kinase inhibitors (TKIs), particularly T315I subtypes. turbinate extract that trabectedin is derived from was Omacetaxine mepesuccinate elicits its antineoplastic shown to have antineoplastic activity as early as 1969 [84], effects through inhibition of protein synthesis. Specifically, separation and characterization of the active molecules was the agent prevents aminoacyl-tRNA from binding the not feasible until the development of sufficiently sensitive ribosomal acceptor site, thereby preventing peptide bond techniques. Further delaying its development was the fact formation at the early stage of protein elongation [79]. In that yields from E. turbinate are extremely low; it takes addition, omacetaxine mepesuccinate inhibits the elonga- 1000 kg of animals to isolate 1 g of trabectedin. It was not tion phase of translation by preventing substrate binding to until the development of synthetic methods of preparation the acceptor site on the 60s ribosome subunit, leading to that actual clinical investigation was feasible. The current the blockade of aminoacyl-tRNA binding and peptide bond supply of trabectedin is based on a semisynthetic process formation [77, 80]. Further, it has been demonstrated that starting from Safracin B, an obtained by fer- the agent blocks protein synthesis by competing with the mentation of the bacterium Pseudomonas fluorescens [85]. amino acid side chains of incoming aminoacyl-tRNAs for Trabectedin is extremely potent, requiring only pico- binding at the A-site of formed ribosomes [81]. Interest- molar to low nanomolar concentrations to initiate cell death ingly, omacetaxine mepesuccinate has shown notable ac- in various cancer types in vitro [86–88]. This notable cy- tivity against leukemic stem cells (LSCs) of CML origin totoxicity is attributed to at least two separate actions; with the agent having a similar inhibitory effect on BCR- DNA alkylation and inhibition of transcription. The agent Natural Product-Based Antineoplastic Agents 2001 binds the minor groove of DNA, showing a preference for sarcoma. This clinical indication has been validated by GG- and GC-rich regions, and then alkylates the exocyclic multicenter phase II trials and a number of noncomparative N-2 on [89, 90]. This alkylation step is dependent phase II trials [99, 100]. In addition, the European Com- on the dehydration of the carbinolamine (also referred to as mission and the FDA have granted orphan drug status to hemiaminal) group found on trabectedin, as this potentiates trabectedin for soft-tissue sarcomas and ovarian carcinoma. the formation of an electrophilic iminium intermediate that The effects of trabectedin against ovarian carcinoma are attracts nucleophilic DNA bases. Two subunits of tra- particularly intriguing; coadministration of trabectedin and bectedin form the primary contacts with DNA, while pegylated liposomal doxorubicin was associated with a another subunit protrudes out of the minor groove [91]. It is significantly longer (6 weeks; p = 0.019) median pro- this subunit that has been associated with the inhibition of gression-free survival than pegylated liposomal doxoru- transcription, as interaction between the subunit and tran- bicin monotherapy in patients with recurrent ovarian scription factors has been observed. Alkylation of DNA carcinoma after progression on platinum-based produces the standard monoalkylating single-strand breaks chemotherapy [101]. In addition, concomitant administra- that proceed to double-strand breaks when the adduct is tion of trabectedin and pegylated liposomal doxorubicin recognized by the transcription-coupled exci- was associated with a relative risk reduction (RRR) in sion repair (TC-NER) complex [92, 93]. These in vitro disease progression or death of 21 % (HR 0.79; 95 % CI observations suggest that patients with BRCA mutations 0.65, 0.96) in comparison to the doxorubicin monotherapy. may receive benefit from trabectedin, as BRCA1 locates These findings are encouraging due to the fact that ovarian DNA damage and attracts the TC-NER complex to repair carcinomas usually portend a poor prognosis (overall DNA breaks, while BRCA2 mediates homologous recom- 5-year survival rate is 44 %, while stage IV invasive bination by loading other proteins to the double-strand ovarian carcinoma has a 5-year survival rate of 17 %) break sites and stalled DNA replication forks [94, 95]. This [102]. The dearth of available treatment options for these postulation has been confirmed in select patients with patients is apparent, warranting further investigation of ovarian carcinoma [93], making trabectedin particularly trabectedin and other novel therapeutic strategies. The attractive for certain subtypes of breast, lung, and ovarian agent has also been investigated in breast carcinoma, lung carcinoma with BRCA mutations. carcinoma, prostate adenocarcinoma [103–105], and vari- Separating trabectedin from other alkylating agents is its ous pediatric sarcomas (Ewing’s sarcoma, and rhab- ability to inhibit the expression of potentially oncogenic domyosarcoma) [106]. transcription factors, including those that code for onco- gene products, c-myb, maf, and myc) and cell-cycle related factors (E2F and SRF), and general transcription factors 7 Finding Novel Utility in Natural Products (SCR, NF-Y, SXR, and Sp1) [96]. Its inhibition of P-gp through Improved Drug Delivery and heat shock protein 70 (Hsp70) expression through NF- Y (as well as SXR, which is also responsible for P-gp The natural products discussed so far are potent antineo- transcription) interaction are of particular intrigue [97, 98], plastic agents, but are all inherently limited by their as both are integral to neoplastic drug resistance. Further, untargeted cytotoxic mechanisms. Although malignant trabectedin has profound activity on the tumor microenvi- cells are preferentially damaged as a result of increased ronment, exerting effects on quiescent tumor-promoting proliferation rates and increased uptake of the given agent, monocytes and macrophages that is atypical of a DNA- normal tissue is also perturbed, preventing higher con- directed alkylating agent [96]. centrations that could elicit more antitumor activity from Trabectedin has linear pharmacokinetics when admin- being administered. Further, there are natural products too istered as a 24-h i.v. infusion as is seen in the standard potent for clinical use because their activity is not specific dosing schedule of 1.5 mg/m2 over 24 h every 3 weeks enough for neoplastic cells to garner any therapeutic ben- [99]. In addition, trabectedin is extensively bound to efit. Finding a delivery system that discriminates between human serum albumin, and the concentration of unbound neoplastic and normal cells, and then transports a cytotoxic trabectedin in the plasma at clinically relevant trabectedin agent across the plasma membrane of aberrant cells to plasma concentrations is in the picomolar range. Drug induce apoptosis would therefore be an ideal situation. As metabolism presides mainly in the liver, with a large it turns out, the idea of preferentially delivering highly number of metabolites being produced by cytochrome cytotoxic natural products to neoplastic tissue has made P450 isozyme 3A4 and to a lesser extent by other cyto- commendable progress in recent years owing to advances chrome P450 isozymes and phase II [99]. in immunotherapy. By using epitopes highly expressed on Trabectedin is approved in Europe (including Russia) the cell surface of malignant cells, investigators have been and South Korea for the treatment of advanced soft-tissue able to develop methods capable of transporting a drug 2002 M. Trendowski payload to the intended target, substantially reducing the thereby potently inhibiting the production of new proteins. toxicity of these agents had they not been conjugated to the Consequently, diphtheria toxin is extraordinarily potent delivery protein. This targeted drug delivery can be with the average human lethal dose being *0.1 lg/kg accomplished through two mechanisms; engineered cyto- [118]. kine proteins and ADCs. Through human innovation, one of the deadliest toxins in nature can be harnessed for cancer therapy once it is 7.1 Engineered Cytokine Proteins given the appropriate delivery system. Denileukin diftitox contains the full-length sequence of IL-2, as well as protein Cytokines are small proteins (*5–20 kDa) important for fragments of diphtheria toxin; 97 amino acids from the various paracrine and autocrine signaling throughout the native part of the toxin containing the disulfide bond are body, but are most associated with their role in the immune removed to increase the half-life and affinity of the com- system. These proteins enable leukocytes to communicate pound to its target receptor [119, 120]. After diphtheria with one another to generate a coordinated, robust, but self- toxin and IL-2 are fused together, the agent seeks out limited response to a target antigen [107, 108]. Cytokines CD25-bearing cells, as CD25 represents the high-affinity themselves have been used for years in chemotherapy a-subunit of the IL-2 receptor. CTCL cells internalize the because of their ability to directly stimulate immune agent via receptor-mediated endocytosis after it binds effector cells and stromal cells at the tumor site, and IL-2R, and is subsequently acidified inside the vesicle. This enhance neoplastic cell recognition by cytotoxic effector process releases diphtheria toxin fragment A into the cells [108–110]. Interleukin-2 (IL-2) is particularly cytoplasm, enabling the internalized fragment A to cat- appealing in hematological malignancies because the IL-2 alyze the transfer of ADP to eEF2. Premature ADP-ribo- receptor (IL-2R) is selectively expressed on activated T sylation of eEF-2 inhibits further protein synthesis, lymphocytes, B cells, and natural killer cells [111]. In a ultimately potentiating apoptosis [121, 122]. normal patient, IL-2R is expressed mostly at a low level in There are limitations to the delivery mechanism by less than 5 % of normal circulating peripheral blood which denileukin diftitox acts. In addition to the expected mononuclear cells [112]. However, many transformed immunosuppression and potential for bacterial infections, leukocytes have a high expression of high or intermediate the agent is known to cause acute hypersensitivity-type affinity IL-2R isoforms, particularly in cutaneous T-cell reactions, asthenia, and / [123]. Further, the lymphoma (CTCL) in which approximately 50 % of cases use of IL-2 as a method to selectively transfer diphtheria express this phenotype as demonstrated by immunohisto- toxin into CTCL cells is inherently limited. IL-2Rs are chemical staining [112, 113]. Therefore, fusing a highly classified into three subtypes based on their affinity for cytotoxic agent to IL-2 may be an effective method by IL-2. Each subtype is composed of a combination of which to target CTCL, while eliciting minimal toxicity to subunits: a (CD25 and p 55), b (CD122 and p 75), and non-hematological tissue. c (CD132 and p 64). Intermediate and high-affinity receptors are composed of IL-2R-b/c (CD122 and CD132) 7.2 Denileukin Diftitox and IL-2R-a/b/c (CD25, CD122, and CD133) subunits, respectively, and both perpetuate internalization and signal Denileukin diftitox is a fusion protein in which the receptor transduction. By contrast, low-affinity receptors consist of binding domain of diphtheria toxin is exchanged for that of the IL-2R-a/c (CD25 and CD132) subunits that bind IL-2, IL-2 (Fig. 1)[114, 115]. Diphtheria toxin is produced but do not cause internalization or activation [112, 124, naturally in the pathogen Corynebacterium diphtheriae, 125]. Therefore, the presence of the b and c (CD122 and and is a single polypeptide chain of 535 amino acids CD132) subunits is essential for sensitivity and internal- consisting of two subunits linked by disulfide bonds, ization of the fusion toxin. If CTCL cells are lacking known as an A–B toxin [116, 117]. The less stable B unit appropriate receptors, denileukin diftitox will be ineffec- binds the cell surface of a target cell, enabling the more tive, paving the way for potential drug resistance. stable A subunit to penetrate the plasma membrane. Once Nevertheless, denileukin diftitox has elicited notable re- internalized, the diphtheria toxin catalyzes the transfer of sponses in patients, and the agent is currently FDA NAD? to a diphthamide residue in eukaryotic elongation approved for persistent or recurrent CTCL in patients factor-2 (eEF2) through ADP-ribosylation, resulting in its expressing the critical CD25 component of IL-2R [18]. deactivation (the same function of endogenous NAD?- Because the expression of IL-2R is vital for the T-cell diphthamide ADP-ribosyltransferase) [118]. In essence, targeting of denileukin diftitox, strategies to upregulate this action halts translation, as eEF2 facilitates the move- IL-2R expression are of particular clinical interest. Reti- ment of the peptidyl tRNA-mRNA complex from the A site noids are a particularly attractive concomitant agent, as they of the ribosome to the P site during protein synthesis; have immunomodulatory function and have been shown to Natural Product-Based Antineoplastic Agents 2003 increase IL-2R expression on T-cells [126, 127]. It has been for stable linker attachment, and still retains high potency demonstrated that both and are in addition to exhibiting water solubility and stability under modulators of high affinity IL-2R expression, as the agents physiological conditions [133]. The ability to conjugate increase the expression of CD25 and CD75 in CTCL cells. MMAE to mABs and potentially other proteins has con- These CTCL cells can be subsequently exposed to deni- siderably increased the utility of auristatins, enabling these leukin diftitox, resulting in a 50–70 % decrease in protein once intolerable agents to be directed towards the intended synthesis [126]. This is particularly exciting because bex- target, thereby dramatically reducing unintended toxicity. arotene is already standard therapy for patients with early- CD30 is an ideal target for ADCs because the antigen is and advanced-stage CTCL. The concomitant use of deni- a tumor necrosis factor receptor that stimulates apoptosis, leukin diftitox and is currently being clinically and is highly expressed in Hodgkin’s lymphoma, systemic investigated [128, 129], and may potentiate a novel avenue anaplastic large cell lymphoma, CTCL, and other selected of chemotherapy for CTCL patients. lymphoid tumors [140, 141], as well as in some non-lym- phoid malignancies including germ cell tumors [141, 142]. 7.3 Antibody-Drug Conjugates Further, cross-reactivity of CD30 on normal tissues is very low, with some expression on activated T and B lympho- Immunoconjugates are a relatively novel class of cytes; this expression is not observed on resting T and B chemotherapeutic agents that are gaining increasing inter- lymphocytes [141]. In addition to the CD30-targeting est because of their efficacy and reduced side effects in mAB, brentuximab vedotin employs a protease-cleavable comparison to some traditional cytotoxic agents. If tradi- linker because protease activity is abundant in lysosomes, tional chemotherapy is equivalated to indiscriminate where ADCs are often directed [133]. In addition, protease bombing, immunoconjugates are roughly analogous to activity is significantly reduced outside of cells owing to modern day missile guidance systems. These agents consist secreted protease inhibitors [141]. MMAE is attached to of three main components; the (mAb; suitable degradable peptides through the built-in N-termi- guidance system), the degradable linker (delivery mecha- nal amine functionality via the self-immolative spacer nism), and the cytotoxic agent (warhead) that can either be (PABC). The spacer is required so that the cleavable a toxin or a radioisotope. While not completely specific to peptide is situated away from MMAE to allow facile pro- neoplastic tissue, immunoconjugates target a given epitope teolysis. Val-Cit (the clevable peptide) is stable in plasma, based on the mAb that is chosen, enabling the use of but is rapidly hydrolyzed via proteolysis by cathepsin in cytotoxic agents that would likely elicit too broad of a lysosomes [143]. Upon peptide cleavage, the PABC group toxicity profile in a typical patient. Although the scope of rapidly fragments, releasing the highly cytotoxic MMAE this review limits commentary to ADCs, radioim- into the cytoplasm. munotherapy has shown efficacy in a variety of cancers, Brentuximab vedotin is currently indicated for Hodg- and the reader would benefit from the following reviews kin’s lymphoma after failure of an autologous stem cell [130–132]. transplant or after failure of two prior multiagent chemotherapeutic regimens in those who are not suit- 7.4 Brentuximab Vedotin able for autologous stem cell transplantation, as well as systemic anaplastic large cell lymphoma. Due to the Brentuximab vedotin is a conjugate of a humanized CD30- prevalence of CD30 in various malignancies, brentuximab targeting mAB and monomethyl auristatin E (MMAE) vedotin is also being investigated in patients with CTCL, linked via a cathepsin cleavable linker (valine-citrulline) other CD30-positive hematological malignancies, and and a para-aminobenzylcarbamate (PABC) spacer (the CD30-positive germ cell tumors [144]. name vedotin refers to MMAE plus its linking structure to the mAB) [133–136]. MMAE is a potent 7.5 Trastuzumab Emtansine derived from the naturally occurring dolastatin 10 that was isolated from Dolabella auricularia (wedge sea hare) in Trastuzumab emtansine is an ADC consisting of the 1987 [137]. Several related molecules have since been humanized HER2/Neu-targeting mAB trastuzumab linked produced through total syntheses, establishing the drug to mertansine (DM1) via SMCC (succinimidyl 4-(N- class auristatin [138]. As with vinca alkaloids, the auris- maleimidomethyl)cyclohexane-1-carboxylate), a heterobi- tatins exert antineoplastic activity by inhibiting tubulin functional crosslinker (Fig. 1)[145, 146]. While trastuzu- polymerization. However. these agents are much more mab is already effective against HER2-positive breast toxic (MMAE is 200 times more potent than vinblastine) carcinomas, esophageal carcinomas, and gastric adenocar- [139], substantially limiting their clinical utility. MMAE cinomas [147, 148], conjugating the mAB with the highly was subsequently developed with a built-in functionality cytotoxic DM1 is an effective method to generate activity 2004 M. Trendowski in refractory or relapsed tumors [149–151]. DM1 refers to Trastuzumab emtansine is currently FDA approved for mertansine, a derivative of maytansine that has been HER2-positive breast carcinoma, HER2-positive meta- chemically modified to have a terminal thiol for conjuga- static gastric adenocarcinomas, and gastroesophageal ade- tion. Maytansine is a macrolide of the ansamycin type nocarcinoma with HER2 overexpression [18]. In the USA, (contains an aromatic moiety bridged by an aliphatic chain) trastuzumab emtansine is typically reserved for the treat- that was originally isolated in 1972 from the flowering ment of HER2-positive metastatic breast carcinoma in plant Maytenus ovatus [152]. Similar to MMAE, may- patients who have been treated previously with trastuzu- tansine is a highly potent microtubule inhibitor that by mab and a taxane (paclitaxel or docetaxel), and who have itself lacks the tumor specificity required to elicit thera- already been treated for metastatic or peutic benefit [153, 154]. It was not until the development developed tumor recurrence within 6 months of adjuvant of ADCs that the extremely cytotoxic potential of may- therapy [166]. This is due in part to the expense of tras- tansine could be harnessed in the form of conjugate-com- tuzumab emtansine treatments, as protocols can cost patible DM1. patients US $9800 a month, or US $94,000 for a typical Trastuzumab is a logical choice for ADC enhancement course of treatment. Since trastuzumab and pertuzumab because its target HER2/Neu is preferentially overex- (another HER2/Neu-targeting mAB) bind different epi- pressed in certain subtypes of breast carcinoma, esophageal topes [167, 168], clinical investigation has also been carcinoma, and gastric adenocarcinoma. In particular, gain- extended to concomitant chemotherapy consisting of both and loss-of-function experiments, as well as immunohis- trastuzumab emtansine and pertuzumab against unre- tochemistry analyses have indicated HER2 amplification as sectable HER2-positive breast carcinoma that is locally a driving event in the onset and progression of as much as advanced or metastatic [169]. 25–30 % of breast carcinomas [155, 156]. Further, this subtype of breast carcinoma is often very aggressive and 7.6 Calicheamicin Conjugated Monoclonal resistant to traditional cytotoxic chemotherapy [157, 158]. Antibodies Due to its apparent oncogene addiction, targeting HER2 positive breast carcinomas through this vital receptor has The cytotoxic agents available for mAB conjugation are become an attractive approach for therapeutic intervention. not limited to highly potent microtubule inhibitors. Perhaps Each molecule of trastuzumab emtansine consists of a the most notable example is calicheamicin conjugated single trastuzumab mAB with several molecules of DM1 mABs. Calicheamicins are a class of enediyne nucleic attached; trastuzumab may be conjugated with up to 8 acid-directed agents that were initially characterized in DM1 molecules (three to four on average) [159, 160]. 1989 from the fermentation broth of the bacterium SMCC contains two reactive functional groups, a succin- Micromonospora echinospora [170]. Since then, imide ester and a maleimide. The succinimide group of calicheamicin c1 and the related enediyne esperamicin SMCC reacts with the free amino group of a lysine residue have become known as two of the most potent antineo- in the trastuzumab molecule, and the maleimide moiety of plastic agents in existence [171]. As is the case with most SMCC links to the free sulfhydryl group of DM1, forming extremely cytotoxic agents, their utility would come not as a covalent bond between the mAB and DM1 [160]. stand-alone agents, but rather as components of epitope- After binding HER2, trastuzumab emtansine gains entry targeting ADCs. The initial attempt to harness the potency to the cellular interior via receptor-mediated endocytosis of calicheamicins was with gemtuzumab ozogamicin, a [161]. Since the non-reducible SMCC linker is stable in humanized CD33-targeting mAB conjugated to a carbo- circulation, as well as in the , hydrate conjugate of N-acetyl-c calicheamicin dimethyl DM1 release occurs only as a result of proteolytic degra- hydrazide via a bifunctional AcBut linker (4-(40- dation of the mAB part of trastuzumab emtansine in the acetylphenoxy)butanoic acid) [172, 173]. Gemtuzumab lysosome. Following release from the lysosome, DM1- ozogamicin was the first FDA-approved ADC, being containing metabolites potently inhibit microtubule approved for the treatment of AML in 2000. Unfortunately, assembly, causing cell-cycle arrest at the G2/M checkpoint, this initial attempt also elicited marked unintended toxicity eventually triggering apoptotic mechanisms [162, 163]. (most notably the potentiation of sinusoidal obstruction Importantly, linkage of DM1 to trastuzumab does not affect syndrome) [174–176], and was withdrawn from the market the binding affinity of trastuzumab to HER2, nor does it in 2010 after a randomized, phase III comparative con- reduce the inherent antineoplastic effects of trastuzumab trolled trial demonstrated that the agent increased patient [162, 164, 165]. Consequently, trastuzumab emtansine death and added no benefit over conventional cancer benefits from the mechanisms of both trastuzumab and therapies [177]. Nevertheless, an additional calicheamicin DM1. conjugated mAb has since been developed (inotuzumab Natural Product-Based Antineoplastic Agents 2005 ozogamicin), and calicheamicin congeners are still being targeting mAB fused to a 38-kDa fragment of Pseu- investigated for their antineoplastic potential. domonas exotoxin A [190]. Moxetumomab pasudotox is The extreme potency of calicheamicins can be traced actually an improved, more active form of a predecessor back to their unique mechanism of action. All calicheam- recombinant immunotoxin, BL22 (also called CAT-3888), icins appear to interact with cellular DNA and initiate which produced complete remission in relapsed/refractory double-strand cleavage by carbon-centered diradical hairy cell leukemia (HCL), but had a \20 % response rate hydrogen abstraction processes [178, 179]. Calicheamicins in chronic lymphoid leukemia (CLL) and ALL, which are bind DNA in the minor groove, wherein they undergo a noted for containing much lower numbers of CD22 [191, reaction analogous to a Bergman cyclization to generate a 192]. Compared with BL22, moxetumomab pasudotox is diradical species. This diradical, 1,4-didehydrobenzene, up to 50-fold more active against CLL and HCL [193]. then abstracts hydrogen atoms from the deoxyribose Pseudomonas exotoxin is a highly cytotoxic product of backbone of DNA, ultimately resulting in irreversible the pathogenic bacterium Pseudomonas aeruginosa [194]. strand scission [180]. The affinity of calicheamicin c1 for With a mechanism very similar to diphtheria toxin, the the minor groove of DNA is due its aryltetrasaccharide exotoxin potently inhibits eEF2 through ADP-ribosylation, domain [181, 182]. Specifically, calicheamicin c1 binds the thereby ceasing further elongation of polypeptides. Crys- DNA minor groove with its aryltetrasaccharide domain in tallographic studies have demonstrated that Pseudomonas an extended conformation spanning TCCT/AGGA seg- exotoxin is made up of three major structural domains; ments of DNA. Calicheamicin c1 then inserts itself in an domain Ia is the cell binding domain, domain Ib contains edgewise manner deep into the minor groove with the no known function, domain II contains a furin site neces- molecule wedged between the walls of the groove [182]. A sary to release domain III from the cell binding domain, range of intermolecular hydrophobic and hydrogen-bond- and domain III contains the ADP-ribosylating activity that ing interactions are also observed, accounting for the inactivates eEF2 [194]. sequence-specific recognition in the complex. In recombinant immunotoxins targeting CD22 (BL22 In addition to gemtuzumab ozogamicin, another and moxetumomab pasudotox), domain Ia of PE is calicheamicin-conjugated humanized mAB, inotuzumab removed and replaced by the Fv portion of a mAB reacting ozogamicin has reached phase III clinical trials. Rather with CD22 [190]. Because BL22 was limited by the lower than targeting CD30, inotuzumab ozogamicin consists of a expression of CD22 in CLL and ALL, the mAB portion of humanized mAb that recognizes the CD22 antigen. Nev- the agent was improved. By mutating three residues in the ertheless, this mAB is still conjugated to N-acetyl-c- heavy chain of the BL22 Fv (residues 100, 100a, and calicheamicin dimethyl hydrazide via the acid labile 4-(40- 100b), investigators were able to increase the affinity of the acetylphenoxy)butanoic acid linker [183]. Since CD22 is a mAB to HCL by about 15-fold and cytotoxicity toward B-lymphocyte-restricted phosphoglycoprotein [184], ino- HCL and CLL cells by up to 50-fold [193]. This tuzumab ozogamicin has been clinically examined in improvement in binding affinity and increased cytotoxicity B-cell leukemias and lymphomas with reversible throm- did not interfere with pharmacokinetics or off-target toxi- bocytopenia as the main toxicity observed. [185, 186]. It city, indicative of superior antineoplastic potential. Mox- should be noted that CD22 may be expressed in certain etumomab pasudotox has produced complete responses lung carcinomas, but this is still inconclusive, as the data against CD22 positive leukemias in the clinical setting, have been conflicting [187, 188]. The ADC has shown demonstrating particular promise with relapsed and notable efficacy against pediatric and adult ALL resistant refractory HCL [195]. Phase III evaluations are currently to traditional cytotoxic chemotherapy [186, 189], and is underway to fully assess the efficacy of moxetumomab indicative of the potential ADCs have in previously unre- pasudotox in comparison to more established therapeutic sponsive cancers. approaches. In addition to its potential with CD22-targeting mAB 7.7 Exotoxin Conjugates Fvs, the pseudomonas exotoxin has also been fused with an anti-mesothelin Fv to create SS1P. Mesothelin is a 40-kDa Antibody/toxin conjugates are not limited to small-mole- cell surface glycoprotein present on some normal cule cytotoxic agents. In a manner similar to denileukin mesothelial cells lining the pleura, peritoneum, and peri- diftitox, mABs have now been successfully conjugated to cardium [196, 197]. However, mesothelin is also highly large protein bacterial exotoxins, thereby increasing the expressed in several malignancies, including epithelial diversity of available immunotoxins. The two most well- mesotheliomas (*100 % of cases), lung carcinomas known examples are moxetumomab pasudotox and SS1P. (*50 % of cases), ovarian carcinomas (*70 % of cases), Moxetumomab pasudotox is a recombinant immuno- and pancreatic/biliary adenocarcinomas (*100 % of toxin composed of the Fv fragment of a fully human CD22- cases) [198–201], thereby being an epitope of particular 2006 M. Trendowski antineoplastic interest. SS1P acts by the same mechanism directed agents available for further preclinical investiga- as moxetumomab pasudotox and is very similar in struc- tion has already been compiled [222]. Therefore, the ture, but is specific for mesothelin-positive cells. This potential of exploiting each currently untargeted compo- epitomizes the utility of immunoconjugates, as the same nent of the cytoskeleton will be briefly highlighted by two highly toxic small molecule or protein can be directed drug classes; cytochalasins and withanolides. towards different targets once linked to an appropriate effector. Demonstrating potent antitumor activity in pre- 8.1 Cytochalasins clinical in vivo models [202–204], SS1P has since pro- gressed towards clinical examination. Owing to its As indicated by both in vitro and in vivo investigation, response in mesothelioma patients during phase I trials microfilaments are vital for the progression of many [205–207], SS1P has gained particular attention in the malignancies, and are therefore a suitable target for treatment of this malignancy. Nevertheless, SS1P has also chemotherapy. One of the most studied drug classes of been clinically investigated in other cancers with high microfilament-directed agents has been the cytochalasins, mesothelin expression, and the agent may gain traction as a which were initially characterized in 1967 as being bio- multipurpose immunotoxin. logical metabolites of the molds Helminthosporium dematiodeum (cytochalasins A and B) and Metarrhizium anisopliae (cytochalasins C and D) [223]. It has since been 8 Natural Products with Mechanisms Not discovered that these mycotoxins potently inhibit actin Currently Seen in the Clinical Setting polymerization, affecting activities ranging from cell motility and adhesion to cytokinesis. Since their initial Although the diversity of natural products currently used in discovery, more than 60 different cytochalasins from sev- the clinical setting is considerably immense, it is not eral species of fungi have been classified into various comprehensive. There are in fact many other potential subgroups based on the size of the macrocyclic ring and the targets inherent to cancer pathology that could be exploited substituent of the perhydroisoindolyl-1-one residue at the through the implementation of novel natural products. One C-3 position [224]. While all cytochalasins demonstrate the target of considerable importance is the cytoskeleton. Not propensity to bind filamentous (F)-actin and block poly- only do many malignant cells have a perturbed cytoskele- merization, only cytochalasins B and D have been exten- ton owing to the effects of dysplasia and subsequent ana- sively studied for their chemotherapeutic potential. Both plasia [208, 209], but the cytoskeleton has also been congeners bind the fast growing barbed end of microfila- indicated in oncogenic signaling and metastatic progres- ments, essentially fulfilling the role of capping proteins that sion [210–212]. While microtubule-directed agents have prevent further actin polymerization once the filament has been a mainstay in traditional cytotoxic chemotherapy, grown to a sufficient length [222]. However, cytochalasin they are inherently limited to one component of the D is more potent than cytochalasin B, reflected by cytoskeleton. The other potential targets, intermediate fil- cytochalasin B being 20-fold less toxic in mice than aments and microfilaments, have remained as elusive cytochalasin D [225], a pharmacological property attrib- clinical prospects. uted to the affinity cytochalasin D also has for globular Cytoskeletal filaments are indeed viable targets to (G)-actin. exploit in chemotherapy. Microfilaments are inherently Although only a handful of laboratories have investi- required for cell motility, cytokinesis, and many other gated the potential of cytochalasins as antineoplastic processes vital for malignant cell stability [213–216]. agents, enough studies have been performed to establish Intermediate filaments such as keratins are often overex- mechanisms by which these agents exert their antineo- pressed in carcinomas as a result of the aberrant effects of plastic effects (Fig. 2). In preclinical mammalian models of associated oncogenes [217, 218], and vimentin has been malignancy, cytochalasins B and D exhibit marked anti- shown to be vital for cell survival in numerous experiments tumor and antimetastatic activity in murine melanomas [219–221]. Although there has yet to be a microfilament- or (B16BL6 and B16F10), lung carcinomas (LA4, Lewis intermediate filament-directed agent approved for clinical Lung, and M109), leukemias (P388 and P388/ADR), and use, nature offers potential solutions to these previously M5076 sarcoma administered intraperitoneally, intra- elusive targets. Through the course of evolution, a con- venously, or subcutaneously [226–239]. In addition, the siderable assortment of small molecules has been devel- pharmacokinetics, tissue distribution, and potential toxici- oped by various organisms that perturb the dynamics of ties of cytochalasin B and liposome-encapsulated actin polymerization or intermediate filament formation, cytochalasin B have been extensively characterized [228– and have demonstrated profound preclinical antineoplastic 231]. The only major toxicity consistently elicited by activity. A comprehensive review of cytoskeletal filament- cytochalasin B is marked immunosuppression, which has Natural Product-Based Antineoplastic Agents 2007

Prevent formaon of the contracle ring and development of the cleavage furrow during cytokinesis, inducing mulnucleaon. Such cells are highly sensive to nucleic acid-directed agents, radiotherapy, and ultrasound. Cell Migraon

Inhibit cell migraon and the secreon of glucosaminidases used to degrade carbohydrates in Inhibit drug efflux by binding ATP-binding cassee the extracellular matrix, potenang marked an- proteins, enhancing the acvity of other metastac acvity. anneoplasc agents.

Fig. 2 Antineoplastic mechanisms of microfilament-disrupting cytochalasins. MW molecular weight been characterized in multiple murine models [230, 231]. [233–242]. Although no potential antineoplastic agent has Nevertheless, this in vivo toxicity can be markedly reduced been identified to specifically target aberrant keratin or either through the administration of human recombinant nestin levels in malignant cells, withanolides have shown IL-2, or liposome encapsulation [228, 229, 231]. Further, promise as a potent type III intermediate filament liposome encapsulated cytochalasin B shows equal, or even inhibitor. superior antitumor activity in M109 lung carcinoma in vivo The most promising clinical prospect of this when compared with the non-encapsulated compound is withaferin A, a steroidal lactone that was initially iso- [228]. It has also been demonstrated by Huang et al. [232] lated from Withania somnifera (winter cherry) in 1965 that a pegylated formulation of cytochalasin D is more [243], and was the first member of the withanolides to be water soluble, accumulates in tumor tissue more efficiently, discovered. Withaferin A has been shown to potently and has a much longer t1/2 than natural cytochalasin D (4 h inhibit a variety of proteins, with the most notable target vs. 10 min), while still retaining the antitumor activity of being vimentin and other type III intermediate filaments the natural compound. [244–248] (Fig. 3). In addition to its affinity for interme- diate filaments, withaferin A has been shown repeatedly to 8.2 Withanolides inhibit angiogenesis [247–251], with potent anti-angio- genic activity being exerted at doses as low as 7 lg/kg/day Along with microfilaments, intermediate filaments are the intraperitoneally in C57BL/6J mice [250]. other component of the cytoskeleton that has yet to be Withaferin A also elicits antineoplastic activity in a exploited in the clinical setting. It has been repeatedly considerable variety of cancer cell lines by directly demonstrated that a variety of malignancies have aberrant inhibiting neoplastic growth, including carcinomas of the or an elevated expression of intermediate filaments keratin breast, head and neck, ovaries, and thyroid, as well as (type I or II), nestin (type VI), and vimentin (type III) glioblastoma multiforme and melanoma [251–255]. Owing 2008 M. Trendowski

Inhibits the formaon of all type 3 intermediate filaments. Elicits anneoplasc effects by downregulang chymotrypc acvity in the proteasome, consequently increasing the amount of ubiquinated proteins and pro-apoptoc proteins targeted by the proteasome. Withaferin A also downregulates important oncoproteins such as survivin.

Reduces neoplasc angiogenesis by inducing endothelial cell death via vimenn modificaon at Has synergisc acvity with sorafenib, and the conserved rod 2B domain, and through inhibion potenally other VEGF small molecule inhibitors or of VEGF. VEGF-targeng mABs.

Fig. 3 Antineoplastic mechanisms of intermediate filament-disrupting withaferin A. MW molecular weight, VEGF vascular endothelial growth factor to its notable anti-angiogenic activity, withaferin A has 9 Conclusion demonstrated significant synergistic effects with the mul- tikinase inhibitor sorafenib [256], suggesting that with- Beginning with the VAMP (vincristine, amethopterin/ aferin A may be a viable supplement for renal cell , 6-, and prednisone) protocol carcinomas, and other malignancies notably affected by the that potentiated long-term survival in pediatric ALL for the inhibition of neoplastic angiogenesis. It should also be first time in medical history [259], natural products have a noted that withaferin A, withalongolide A (a 19-hydroxy long history as antineoplastic agents. They have repeatedly derivative of withaferin A), and several other closely added to the mechanisms of action available to practicing related congeners may influence signaling regulated by the clinicians, as well as inspire semi-synthetic derivations that PI3K/AKT/mTOR pathway. These congeners suppress demonstrate improved clinical utility. Although sometimes RET and Akt phosphorylation and protein expression, as archaic by their less than ideal discrimination of neoplastic well as inhibit mTOR activity, the translational activity of over normal tissue, these agents are still highly imple- 4EBP1, and protein synthesis mediated by p70S6 kinase mented in many forms of chemotherapy. In an age where activation in neoplastic cells [patient-derived medullary clinicians are becoming ever more sophisticated in their thyroid carcinoma cells, as well as human (U87 and U251) ability to target solid tumors and disseminated cancers with and murine (GL26) glioblastoma cells] in vitro [257, 258]. small-molecule inhibitors and the rapidly expanding field These data indicate a potential synergistic relationship of immunotherapy, we should not lose sight of the between withanolides and mTOR inhibitors currently used importance of natural products for the treatment of cancer. in the clinical setting, and warrant further preclinical Epitomized by ADCs, natural products previously deemed investigation. too potent to elicit therapeutic benefit can now be Natural Product-Based Antineoplastic Agents 2009 conjugated to an appropriate protein delivery system, 4. Gordaliza M. Natural products as leads to anticancer drugs. Clin thereby delivering highly cytotoxic and specific treatments Transl Oncol. 2007;9(12):767–76. 5. Demain AL, Vaishnav P. Natural products for cancer to neoplastic tissue. This is an important lesson, as there are chemotherapy. Microb Biotechnol. 2011;4(6):687–99. currently many cytotoxic mechanisms of action employed 6. Newman DJ, Cragg GM. Natural products as sources of new in nature that are not currently used in the clinical setting, drugs over the 30 years from 1981 to 2010. J Nat Prod. such as the microfilament-targeting cytochalasins, or the 2012;75(3):311–35. 7. Hirata Y, Uemura D. Halichondrins—antitumor polyether intermediate filament-targeting withanolides. macrolides from a marine sponge. Pure Appl Chem. While precision medicine has captivated the field of 1986;58(5):701–10. oncology in recent years and is gaining an increasing 8. Towle MJ, Salvato KA, Budrow J, Wels BF, Kuznetsov G, Aalfs foothold in the clinic, we should also not discourage the KK, Welsh S, Zheng W, Seletsky BM, Palme MH, Habgood GJ, Singer LA, Dipietro LV, Wang Y, Chen JJ, Quincy DA, Davis continual investigation of natural products as antineoplastic A, Yoshimatsu K, Kishi Y, Yu MJ, Littlefield BA. In vitro and agents. As demonstrated by current protocols, their unique in vivo anticancer activities of synthetic macrocyclic ketone mechanisms can be coupled with synthetic inhibitors or analogues of halichondrin B. Cancer Res. 2001;61(3):1013–21. genetically modified proteins to create effective first-line 9. Jain S, Vahdat LT. Eribulin mesylate. Clin Cancer Res. 2011;17(21):6615–22. therapies. Further, some derivatives of natural products 10. Cortes J, Montero AJ, Glu¨ck S. Eribulin mesylate, a novel such as the rapamycin analogs have the potential to be used microtubule inhibitor in the treatment of breast cancer. Cancer in select cancers that demonstrate specific aberrant signal- Treat Rev. 2012;38(2):143–51. ing cascades. Nature has already done the hard work 11. Jordan MA, Kamath K, Manna T, Okouneva T, Miller HP, Davis C, Littlefield BA, Wilson L. The primary antimitotic mechanism through years of evolutionary pressures. Now, modern of action of the synthetic halichondrin E7389 is suppression of medicine can harness these complex, but often synthetically microtubule growth. Mol Cancer Ther. 2005;4(7):1086–95. reproducible molecules, to develop ever more comprehen- 12. Alday PH, Correia JJ. Macromolecular interaction of halichon- sive and effective treatment modalities. As demonstrated by drin B analogues eribulin (E7389) and ER-076349 with tubulin by analytical ultracentrifugation. Biochemistry. 2009;48(33): the countless lives they have prolonged or even saved, 7927–38. natural products have, are still, and will continue to function 13. Smith JA, Wilson L, Azarenko O, Zhu X, Lewis BM, Littlefield as vital components of cancer chemotherapy. BA, Jordan MA. Eribulin binds at microtubule ends to a single site on tubulin to suppress dynamic instability. Biochemistry. Acknowledgments The author extends his sincerest gratitude to all 2010;49(6):1331–7. of the investigators who have been involved in the development of 14. Okouneva T, Azarenko O, Wilson L, Littlefield BA, Jordan MA. natural product-based antineoplastic agents, including his mentor, Dr. Inhibition of centromere dynamics by eribulin (E7389) during Thomas Fondy of Syracuse University. In addition, the author thanks mitotic metaphase. Mol Cancer Ther. 2008;7(7):2003–11. the two anonymous reviewers whose suggestions helped improve the 15. Ortega Vanesa, Corte´s Javier. Potential clinical applications of quality of the article. halichondrins in breast cancer and other neoplasms. Breast Cancer (Dove Med Press). 2012;4:9–19. Compliance with Ethical Standards 16. Swami U, Chaudhary I, Ghalib MH, Goel S. Eribulin: a review of preclinical and clinical studies. Crit Rev Oncol Hematol. 2012;81(2):163–84. Funding information The author thanks Syracuse University for 17. Shetty N, Gupta S. Eribulin drug review. South Asian J Cancer. providing funds for research on microfilament-directed agents. 2014;3(1):57–9. 18. Abraham J, Gulley JL, Allegra CJ. The Bethesda handbook of Conflict of interest The author declares no conflicts of interest. clinical oncology, 4th ed. Philadelphia: Lipincott Williams & Wilkins; 2014. Open Access This article is distributed under the terms of the 19. Cortes J, O’Shaughnessy J, Loesch D, Blum JL, Vahdat LT, Creative Commons Attribution-NonCommercial 4.0 International Petrakova K, Chollet P, Manikas A, Die´ras V, Delozier T, License (http://creativecommons.org/licenses/by-nc/4.0/), which per- Vladimirov V, Cardoso F, Koh H, Bougnoux P, Dutcus CE, mits any noncommercial use, distribution, and reproduction in any Seegobin S, Mir D, Meneses N, Wanders J, Twelves C. Eribulin medium, provided you give appropriate credit to the original monotherapy versus treatment of physician’s choice in patients author(s) and the source, provide a link to the Creative Commons with (EMBRACE): a phase 3 open- license, and indicate if changes were made. label randomised study. Lancet. 2011;377(9769):914–23. 20. Scarpace SL. Eribulin mesylate (E7389): review of efficacy and tolerability in breast, pancreatic, head and neck, and non-small cell . Clin Ther. 2012;34(7):1467–73. 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